3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P35 COMPARISON OF PROTEOME AND
METAbOLOME ChANGES IN STRESSED
yEAST STRAINS rHODOTOrulA gluTiNis
AND rHODOTOrulA rubrA
AnDREA HALIEnOVá a , IVAnA MáROVá a ,
ZBYněK ZDRáHAL b , HAnA KOnEČná b , MARTInA
ČARnECKá a , KATEřInA PAřILOVá a and EMíLIA
BREIEROVá c
a Department of Food Chemistry and Biotechnology, Faculty
of Chemistry, Brno University of Technology, Purkyňova 118,
612 00 Brno, Czech republic,
b Laboratory of Functional Genomics and Proteomics, Faculty
of Science, Masaryk University of Brno, Czech Republic,
c Institute of Chemistry, Slovak Academy of Sciences, Dúbravská
cesta 9, 845 38 Bratislava, Slovak Republic,
halienova@fch.vutbr.cz
Introduction
Exogenous stress and other environmental factors can
induce many changes in cell composition. Some of these
changes involved in stress response may lead to over- or
underexpression of cell proteins. Identification of metabolic
markers characteristic for certain events provides important
insight into the metabolism control. Additionally, this information
could be used to the biotechnological production of
some industrially significant metabolites.
Carotenogenic yeasts produce high amount of lipidic
compounds. Carotenoids are membrane-bound lipid-soluble
pigments, which can act as effective antioxidants and scavenge
singlet oxygen. In red yeasts they probably act as adaptive
and/or protecive mechanism against exogenous oxidative
stress and UV-irradiation. Carotenoids are produced by a specific
branch of common isoprenoid pathway and accumulated
in particular cell organelles.
In this work, protein and metabolic profiles of salt- and
peroxide- stressed carotenogenic yeasts of the genus Rhodotorula
were analyzed. Yeast cells Rhodotorula glutinis CCY
20-2-26 and Rhodotorula rubra CCY 20-7-29 were cultivated
in glucose medium in presence of 2–5% naCl and 2–5 mM
hydrogen peroxide. In yeast cells carotenoids and ergosterol
as specific stress metabolites were measured using HPLC/
DAD. Proteins were separated by 1D and 2D electrophoresis.
Some spots were identified by LC/MS/MS.
Materials and Methods
S t r a i n s
Industrial yeasts Rhodotorula glutinis CCY 20-2-26
and Rhodotorula rubra CCY 20-7-29 were used as tested
strains. As a comparative strain Saccharomyces cerevisiae
CCY 21-4-88 was used. Red yeasts were cultivated on glucose
medium aerobically at 28 °C. Exogenous stress was
induced by 2–5 mM peroxide and 2–5% naCl.
s647
C e l l F r a c t i n a t i o n
Protein fractions of red yeast cells were obtained by
gradually separation using combination of chemical (naOH
and detergents) and mechanical (glass beads; 100 µm) lysis.
Protein fraction for 2D analysis was isolated mainly from
lyophilized cells.
C a r o t e n o i d A n a l y s i s
Levels of carotenoids – lycopene, beta-carotene, torulen
and phytoene were analyzed using HPLC/MS. Ergosterol
was analyzed by RP-HPLC (280 nm).
1 D G e l E l e c t r o p h o r e s i s
1D PAGE-SDS electrophoresis of proteins was carried
out by common procedure using 10% and 12.5% polyacrylamide
gels. Proteins were staining by Coomassie Blue and by
silver staining. For comparison, microfluidic technique using
1D Experion system (BioRad) and P 260 chips was used for
yeast protein analysis too.
2 D G e l E l e c t r o p h o r e s i s a n d L C -
M S / M S
2D electrophoresis of proteins was optimized in cooperation
with Laboratory of Functional Genomics and Proteomics,
Faculty of Science, Masaryk University of Brno.
2D gels were obtained using protein preparatives isolated
from lyophilized cells. After optimization of separation conditions
proteomes from stressed R. glutinis and R. rubra cells
were isolated, lyophilized and analyzed. Quantitative analysis
was done using BioRad Laboratories 2D software. Identification
of some spots was done using LC-MS/MS.
Results
Proteins from red yeast species were isolated with cell
lysis combined with mechanical disintegration and protein
profiles were obtained by 1D and 2D electrophoresis. Meta-
Fig. 1. 1D protein profiles – stressed r. rubra (NaOh);
lane 1 – standard 6, 2 – proteome from lyofilized cells, 3 – proteome
from non-lyofilized cells, 4 – proteome 2% NaCl, lyo,
5 – 2 % NaCl, non-lyo, 6–7 – 5 % NaCl, 8–9 – 2 mM hydrogen
peroxide, 10–11 – 5 mM peroxide; 12, 13 – standards 4, 5